1. Field of the Invention
This invention resides in the field of hydrolases (i.e., hydrolytic enzymes), and in particular, methods and devices for the detection of hydrolase activity in a sample or specimen and the diagnosis of disease based on the detection of hydrolase activity. A specific area of interest of this invention is the detection of trichomoniasis in female subjects by assaying for the presence of enzymatically active trichomonal hydrolases in a vaginal fluid specimen.
All literature cited in this specification, including patents, technical articles and books, are incorporated by reference in their entirety.
2. Description of the Prior Art
Trichomoniasis is a clinically important Sexually Transmitted Disease (STD) caused by the protozoan Trichomonas vaginalis. In 1955, the World Heath Organization estimated approximately 170 million new cases of trichomoniasis would arise annually among adults worldwide, with higher prevalence and incidence rates in both developing and industrialized countries than for any other sexually transmitted disease. Trichomoniasis is frequently undetected because most infected men and approximately 50% of infected women are asymptomatic. Trichomoniasis in women may cause discomfort or a foul smelling vaginal discharge, and can be associated with adverse clinical sequelae. Trichomoniasis can increase the risk the human immunodeficiency virus (HIV) transmission and infection (Laga et al., AIDS 7(1):95–102 (1993) and WHO Press Release WHO/64 (1995)).
Trichomonads produce specific hydrolases called proteinases that hydrolyze proteins including, but not limited to, IgG, IgM, and IgA antibodies (Provenzano and Alderete, Infect. Immun. 63(9):3388–3395 (1995)). These proteinases also damage secretory leukocyte hydrolase inhibitor (SLPI), a protective factor normally present in vaginal fluid that inhibits HIV viral entry into human monocytic cells (Draper et al., J. Infect. Dis. 178(3):815–819 (1998)). Trichomonas vaginalis (T. vaginalis) is also thought to play a role in promoting cervical cancer (Yap et al., Genitourin. Med. 71(6):402–404 (1995)). Pregnant women infected with T. vaginalis at mid-gestation are more likely to have a low birth weight infant or to deliver preterm (Cotch et al., Sex. Transm. Dis. 24:353–360 (1997)).
The most common method for diagnosing trichomoniasis is wet mount microscopy, a microscopic examination of vaginal fluid specimens for T vaginalis. This is a labor-intensive method which requires a microscope and a skilled technician, and fails to detect approximately half of the infected women (Baron et al., Laboratory Diagnosis of Female Genital Tract Infections, Cumitech 17A, American Society for Microbiology (1993)). Trichomoniasis can be diagnosed with high sensitivity by culturing vaginal fluid specimens, but this method requires culture media and long-term growth in a controlled-temperature incubator, in addition to the use of a microscope and a skilled technician. Due to the labor involved, the expense of culture media and supplies, the training required, and the delay of up to a week to detect trichomonal growth, few clinics or laboratories routinely use culture to diagnose trichomoniasis.
A diagnostic test based on DNA amplification and detection has recently been developed by Lawing et al., J. Clin. Microbiol. 38(10):3585–3588 (2000). This test is sensitive but, like culture, not rapid enough to produce the results during the patient's visit to the clinic. Due to the cost of the DNA-based test kits and the training and laboratory equipment required to perform these tests, this methodology is not widely used in clinics or in medical practice in general, particularly in the parts of the world where the need for a trichomoniasis test is greatest.
Thus, to date there is no rapid, accurate, cost-effective and simple method or test device for point-of-care diagnosis of trichomoniasis. Numerous studies have demonstrated that T. vaginalis produces a variety of hydrolases; see for example Garber et al., Can. J. Microbiol. 35:903–909 (1989). Two studies have demonstrated that vaginal fluid specimens from women with trichomoniasis contain detectable levels of hydrolases that disappear after infected women are treated and cured of the infection (Alderete et al., Genitourin. Med. 67(6):469 (1991), and Garber and Lemchuck-Favel, Parasitol. Res. 80(5):361–365 (1994)). Unfortunately, the laboratory-based hydrolase detection methods used in the studies reported by these authors required equipment, skill and training and were labor-intensive and slow, so that the results were obtained only after several hours. For example, the method used by Aldrete et al. for detecting trichomonal hydrolases involved a four-step process to produce gelatin-acrylamide zymograms: first, trichomonal hydrolases were electrophoretically concentrated into discrete bands in a sheet of polyacrylamide gel; second, the hydrolases were allowed to digest gelatin which had been immobilized within the polyacrylamide gel; third, the gel was stained with a general protein stain; and fourth, the gel was destained in a lengthy washing process which eventually revealed the hydrolase-digested gelatin which appeared as clear bands on the darkly stained background gel (the zymogram). Trichomonal hydrolases were detected in the Garber et al. study by polyacrylamide gel electrophoresis followed by immunoblotting, a similarly complex procedure that involved the use of rabbit antibodies to visualize a specific hydrolase band. Both methods require a high level of expertise and costly equipment and take many hours to complete, and are therefore unsuitable for point-of-care testing.
The zymogram procedure described above can also be performed by using synthetic fluorogenic substrates to detect the trichomonal hydrolases once the hydrolases have been separated by gel electrophoresis. These substrates typically contain one or more amino acids linked to a fluorogenic reporter group that becomes fluorescent only after it is enzymatically cleaved from the peptide group by a hydrolase. Unfortunately, this procedure still entails the unwieldy polyacrylamide gel electrophoresis step prior to testing for hydrolase activity with the fluorogenic substrates. Moreover, hydrolysis of the substrate can be observed only by examining the electrophoretic gels for bands that fluoresce under ultraviolet light. Both the gelatin-digestion method and the fluorogenic substrate method were utilized in a study of intracellular and secreted T. vaginalis hydrolases reported by North et al., Mol. Biochem. Parasitol. 39:183 (1990). Several fluorogenic substrates were identified which could be used to detect trichomonal hydrolases. Unfortunately, the methods used in this study are impractical for a point-of-care clinical diagnostic test, since gel electrophoresis is slow and cumbersome and observation of fluorescence requires instrumentation or a darkroom and ultraviolet illuminator. The difficulty is that vaginal fluid contains many different hydrolases secreted by a variety of sources, including bacteria, which are present at extremely high levels, white blood cells, vaginal epithelial cells, and others. Each method relies on electrophoretic separation to achieve selective detection of the trichomonal hydrolases. Without clectrophoretic separation, it could not be determined if the hydrolytically active bands were derived from trichomonads or from some other source of hydrolytic activity in a vaginal fluid specimen.
A trichomoniasis test is therefore needed that can be performed by attending clinicians quickly, simply, inexpensively and accurately while the patient is still present. It would be particularly beneficial to be able to perform the test with a disposable device that is inexpensive and easy to use and one that rapidly produces accurate results.